Pharmaceutical composition for enhancing antitumor effect by immune checkpoint inhibitor

ABSTRACT

The object of the invention is to provide a pharmaceutical composition for enhancing the antitumor effect by an immune checkpoint inhibitor. Provided is a pharmaceutical composition for enhancing the antitumor effect by an immune checkpoint inhibitor comprising 5-aminolevulinic acids (ALAs) as the active ingredient.

TECHNICAL FIELD

The present invention relates to a novel use of 5-aminolevulinic acid(hereinafter also simply referred to as “ALA”) or a derivative thereof,specifically to the use of 5-aminolevulinic acid or a derivative thereoffor enhancing the antitumor effect by an immune checkpoint inhibitor.

BACKGROUND ART

Tumors that had emerged in the human body are removed by the ordinarilyinherent immune mechanism before they proliferate to an extent that theyhave harmful effects on the human body. The following phenomenon isknown as this mechanism. Namely, a protein (tumor antigen) specific tocancer cells binds to a protein called class I MHC molecule in cancercells, and is presented on the cell surface. When this class I MHC andthe tumor antigen binds to the TCR (T Cell Receptor) protein on thesurface of T cells which are responsible for immunity, the T cellrecognizes this cell as a cancer cell. The T cell which recognized thecancer cell initiates proliferation, and damages the cancer cellpresenting the tumor antigen.

However, it is known that when the cancer cell was expressing a membraneprotein called PD-L1 together with class I MHC, by binding with amembrane protein called PD-1 which is being expressed in the T cell, theactivation of the above T cell is suppressed. Because this cancer cellwhich has acquired the immunological escape mechanism which is normallyfor suppressing excess immunity continues to proliferate, inhibition ofthe binding between PD-L1 and PD-1 has been focused as the target for anew anticancer agent (Patent Literature 1). Note that PD-L1 is alsoexpressed in “professional antigen-presenting cells” such as dendriticcells which specialize in presenting antigens to T cells. In recentyears, anti-PD-1 antibodies nivolumab (Opdivo®) and pembrolizumab(Keytruda®) have been developed and put to practical use as anticanceragents, and anti-PD-L1 antibodies atezolizumab, durvalumab, avelumab(Bavencio™), and the like have also been developed and put to practicaluse.

On the other hand, in the antigen presentation of cancer cells (orprofessional antigen-presenting cells) to T cells, other mechanisms tosuppress immune reaction (immune checkpoints) are also known, arepresentative of which is a combination of the B7 family molecule onthe antigen-presenting side and CTLA4 on the T cell side, andpharmaceuticals such as ipilimumab (Yervoy®) and tremelimumab have beendeveloped. In addition, CD137L-CD137, MHC-LAG-3/KIR, CD48-CD244,GAL9-TIM3, HVEM-BTLA/CD160, CD40L-CD40, OX40L-OX40, GITRL-GITR, and thelike are known (in all of which the former is the molecule on theantigen-presenting side) (Non-Patent Literatures 1 and 2).

Recently, there has been a report that the antitumor effect by ananti-PD-1 antibody is related to the generation of reactive oxygen in Tcells, and that the said antitumor effect is enhanced by intracellularsignal transduction and mitochondrial activation by addition of areactive oxygen generator at a low concentration. Moreover, it isreported at the same time that the antitumor effect of an anti-PD-1antibody is also enhanced in pharmaceuticals that mimics thisintracellular signal transduction (Non-Patent Literature 3).

The present inventors have found that an ALA or a derivative thereof iseffective for cancer therapy by a presumed mechanism which enhances theheme or cytochrome in the mitochondria of a cancer cell or a cell almostbecoming a cancer cell having formed an abnormality in the nucleus,improves mitochondrial activities such as the electron transport chainand the TCA cycle, and calls up the Bax and Bak systems to cause acaspase IX-type apoptosis when there is an abnormality in the nucleusthat cannot be restored (Patent Literature 2).

On the other hand, ALA together with an arbitrary sodium ferrous citrate(SFC) becomes a heme in the body, and this heme is degradated by anenzyme called a heme oxygenase 1 (HO-1) to change into bilirubin andcarbon monoxide (Non-Patent Literature 4). It is known that thisbilirubin and carbon monoxide has a high antioxidant action and candirectly/indirectly erase reactive oxygen species (ROS) (Non-PatentLiterature 5)

Moreover, ALA, together with an arbitrary SFC, is known to have animmunological tolerance effect which suppresses immunity. This mechanismis thought to be where, in a dendritic cell which is theantigen-presenting cell, the above-described HO-1 (or bilirubin andcarbon monoxide) changes (differentiates) the dendritic cell into a cellcalled a tolerogenic dendritic cell. A tolerogenic cell is known tohighly express PD-L1 and to perform specific immunological tolerance(immunosuppression) on the antigen presented on the T cell, and in fact,a rise in the mRNA of PD-L1 has been observed in a cell that isrecognized as a dendritic cell by administration of ALA and SFC(Non-Patent Literature 6).

CITATION LIST

-   [Patent Literature 1] JP Patent No. 5885764-   [Patent Literature 2] JP Patent No. 5611548-   [Non-Patent Literature 1] Nat Rev Cancer. 2012 Mar. 22; 12(4):    252-64.-   [Non-Patent Literature 2] Ann Transl Med. 2015 October; 3(18): 267.-   [Non-Patent Literature 3] Proc Natl Acad Sci USA. 2017 Jan. 31;    114(5): E761-E770.-   [Non-Patent Literature 4] Am J Physiol Renal Physiol. 2013 Oct. 15;    305(8)F1149-57-   [Non-Patent Literature 5] Am J Physiol Cell Physiol. 2015 Apr. 15;    308(8)C665-72.-   [Non-Patent Literature 6] J Heart Lung Transplant. 2015 February;    34(2): 254-63.

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

As described above, cancer therapy agents that comprise an anti-PD-1antibody or an anti-PD-L1 antibody as the active ingredient have beencreated, and further, a combination therapy method of these cancertherapy agents and preexisting pharmaceuticals has been drawingattention. In fact, a clinical trial of the combination ofpharmaceuticals that mimic the above signal transduction (lipid-loweringdrug bezafibrate that activates the transcription factor PPARs) and ananti-PD-1 antibody has been planned. Namely, there is a demand for thesearch or creation of a medicament or a pharmaceutical composition whichhas high correlation with immune checkpoint inhibitors such as ananti-PD-1 antibody or an anti-PD-L1 antibody and which may enhance theantitumor effect by an immune checkpoint inhibitor.

Accordingly, the object of the present invention is to provide apharmaceutical composition for enhancing the antitumor effect by immunecheckpoint inhibitors such as an anti-PD-1 antibody or an anti-PD-L1antibody.

Means for Solving the Problems

As a result of repeated extensive investigation in order to solve theabove problem, the present inventors found that 5-aminolevulinic acid(ALA) can significantly enhance the antitumor effect by an immunecheckpoint inhibitor. As above, although ALA itself may be useful forcancer therapy, in consideration of the high antioxidant action bybilirubin and carbon monoxide (CO) resulting from administration of ALAor the immunological tolerance effect by ALA, when ALA is administeredduring cancer therapy by immune check inhibitors such as an anti-PD-1antibody or an anti-PD-L1 antibody, it was rather thought that theeffect of the immune checkpoint inhibitor will be counteracted due toALA causing increase of PD-L1-expressing dendritic cells or reactiveoxygen in T cells being removed by bilirubin and CO, and thus thisresult was surprising.

Namely, the present invention encompasses the following characteristics:

[1] A pharmaceutical composition for enhancing the antitumor effect ofan immune checkpoint inhibitor, comprising the compound shown by thefollowing Formula (I):

[Chemical Formula 1]

R¹—NHCH₂COCH₂CH₂COOR²  (I),

(wherein R¹ represents a hydrogen atom or an acyl group, and R²represents a hydrogen atom, a linear or branched alkyl group, acycloalkyl group, an aryl group, or an aralkyl group) or a salt or anester thereof.

[2] The pharmaceutical composition according to [1], wherein said immunecheckpoint inhibitor is at least one selected from the group consistingof an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4antibody, an anti-B7 antibody, an anti-C27 antibody, an anti-KIRantibody, an IDO inhibitor, an anti-CD137 antibody, and an anti-TIM3antibody.

[3] The pharmaceutical composition according to [2], characterized inthat the immune checkpoint inhibitor is an anti-PD-L1 antibody or ananti-PD-1 antibody.

[4] The pharmaceutical composition according to [1], wherein said immunecheckpoint inhibitor is selected from the group consisting ofatezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab,pidilizumab, BMS-936559, ipilimumab, tremelimumab, enoblituzumab,varlilumab, lirilumab, epacadostat, utomilumab, urelumab, and TSR-022.

[5] The pharmaceutical composition according to any of [1]-[4],characterized in that it is administered at the same time or atdifferent times as the immune checkpoint inhibitor.

[6] The pharmaceutical composition according to any of [1]-[6],characterized in that:

R¹ is selected from the group consisting of a hydrogen atom, an alkanoylgroup having 1-8 carbons, and an aroyl group having 7-14 carbons, and

R² is selected from the group consisting of a hydrogen atom, a linear orbranched alkyl group having 1-8 carbons, a cycloalkyl group having 3-8carbons, an aryl group having 6-14 carbons, and an aralkyl group having7-15 carbons.

[7] The pharmaceutical composition according to any of [1]-[7], whereinR¹ and R² are hydrogen atoms.

[8] The pharmaceutical composition according to any of [1]-[8], whichfurther contains one, or two or more types of metal-containingcompounds.

[9] The pharmaceutical composition according to [8], wherein themetal-containing compound is a compound containing iron, magnesium,zinc, nickel, vanadium, copper, chromium, molybdenum, or cobalt.

[10] The pharmaceutical composition according to [9], wherein themetal-containing compound is a compound containing iron, magnesium, orzinc.

[11] The pharmaceutical composition according to [10], wherein themetal-containing compound is a compound containing iron.

[12] The pharmaceutical composition according to [11], wherein saidcompound containing iron is sodium ferrous citrate.

[13] The use of the compound shown by the following Formula (I):

[Chemical Formula 2]

R¹—NHCH₂COCH₂CH₂COOR²  (I),

(wherein R¹ represents a hydrogen atom or an acyl group, and R²represents a hydrogen atom, a linear or branched alkyl group, acycloalkyl group, an aryl group, or an aralkyl group) or a salt or anester thereof in the production of a medicament for enhancing theantitumor effect of an immune checkpoint inhibitor.

[14] A method for enhancing the antitumor effect of an immune checkpointinhibitor, comprising administering the compound shown by the followingFormula (I):

[Chemical Formula 3]

R¹—NHCH₂COCH₂CH₂COOR²  (I),

(wherein R¹ represents a hydrogen atom or an acyl group, and R²represents a hydrogen atom, a linear or branched alkyl group, acycloalkyl group, an aryl group, or an aralkyl group)or a salt or an ester thereof to a subject to which an immune checkpointinhibitor is administered or being administered.

Note that an invention of any combination of one or more characteristicsof the present invention described above is also encompassed by thescope of the present invention.

Effects of the Invention

According to the present invention, a pharmaceutical composition forenhancing the antitumor effect by an immune checkpoint inhibitor isprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the administration schedule of each test agent in the testof Example 1.

FIG. 2 shows the comparison among the non-treated group, the anti-PD-L1antibody administration group, the anti-PD-L1 antibody+ALAadministration group, and the anti-PD-L1 antibody+ALA+SFC administrationgroup in the tumor volume after tumor inoculation. * shows that p<0.05by t-test when the variances of the two populations are not equal.

FIG. 3 shows the administration schedule of each test agent in the testof Example 2.

FIG. 4 shows the comparison among the non-treated group, the anti-PD-1antibody administration group, and the anti-PD-1 antibody+ALA+SFCadministration group in the tumor volume after tumor inoculation.

DESCRIPTION OF EMBODIMENTS

The present invention will now be described in detail.

The present invention relates to a pharmaceutical composition forenhancing the antitumor effect by an immune checkpoint inhibitor(hereinafter also referred to as “the pharmaceutical composition of thepresent invention”).

An “immune checkpoint inhibitor” is an anticancer agent that suppressesthe proliferation of cancer by binding to an immune checkpoint moleculewhich suppresses T cell activity due to presentation of antigen, andinhibiting its signal transduction. Immune checkpoint molecules mayinclude both receptors and ligands which function as immune checkpoints.

In one embodiment of the present invention, an “immune checkpointinhibitor” includes, but is not limited to, e.g. any antibody orcompound that can inhibit the binding or interaction between PD-1L andPD-1, the binding or interaction between CD80/CD86 and CTLA4, thebinding or interaction between CD137L and CD137, the binding orinteraction between MHC and LAG-3/KIR, the binding or interactionbetween CD48 and CD244, the binding or interaction between GAL9 andTIM3, the binding or interaction between HVEM and BTLA/CD160, thebinding or interaction between CD40L and CD40, the binding orinteraction between OX40L and OX40, and the binding or interactionbetween GITRL and GITR.

In another embodiment of the present invention, an “immune checkpointinhibitor” is selected from the group consisting of, but is not limitedto, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4antibody, an anti-B7 antibody, an anti-C27 antibody, an anti-KIRantibody, an IDO inhibitor, an anti-CD137 antibody, and an anti-TIM3antibody.

In further another embodiment of the present invention, an “immunecheckpoint inhibitor” is selected from, but is not limited to,atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab,pidilizumab, BMS-936559, ipilimumab, tremelimumab, enoblituzumab,varlilumab, lirilumab, epacadostat, utomilumab, urelumab, and TSR-022.

In further another embodiment of the present invention, an “immunecheckpoint inhibitor” is an anti-PD-L1 antibody or an anti-PD-1antibody.

The pharmaceutical composition of the present invention is characterizedin that it has ALA or a derivative, salt, or ester thereof (hereinafteralso simply referred to as “ALAs”) as the active ingredient.

An ALA as used herein means 5-aminolevulinic acid. ALA is also referredto as 8-aminolevulinic acid, and is a type of amino acid.

The compound shown by the following Formula (I) can be exemplified as anALA derivative. In Formula (I), R¹ represents a hydrogen atom or an acylgroup, and R² represents a hydrogen atom, a linear or branched alkylgroup, a cycloalkyl group, an aryl group, or an aralkyl group. Note thatin Formula (I), ALA corresponds to when R¹ and R² are hydrogen atoms.

[Chemical Formula 4]

R¹—NHCH₂COCH₂CH₂COOR²  (I),

ALAs may act as an active ingredient in vivo in the form of the ALA ofFormula (I) or a derivative thereof, and can also be administered as aprodrug (precursor) that is degradated by an in vivo enzyme.

The acyl group in R¹ of Formula (I) can include a linear or branchedalkanoyl group having 1-8 carbons such as formyl, acetyl, propionyl,butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl,and benzylcarbonyl groups, or an aroyl group having 7-14 carbons such asbenzoyl, 1-naphthoyl, 2-naphthoyl groups.

The alkyl group inR² of Formula (I) can include a linear or branchedalkyl group having 1-8 carbons such as methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl,hexyl, heptyl, and octyl groups.

The cycloalkyl group in R² of Formula (I) can include a cycloalkyl grouphaving 3-8 carbons which may be saturated or have partially unsaturatedbonds, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclododecyl, and 1-cyclohexenyl groups.

The aryl group in R² of Formula (I) can include an aryl group having6-14 carbons such as phenyl, naphthyl, anthryl, and phenanthryl groups.

The aralkyl group in R² of Formula (I) can be exemplified with the samearyl groups as above as the aryl moiety and the same alkyl groups asabove as the alkyl moiety, and can specifically include an aralkyl grouphaving 7-15 carbons such as benzyl, phenethyl, phenylpropyl,phenylbutyl, benzhydryl, trityl, naphthylmethyl, and naphthylethylgroups.

Preferred ALA derivative include compounds where R¹ is a formyl group,an acetyl group, a propionyl group, a butyryl group, and the like.Moreover, preferred ALA derivatives also include compounds where theabove R² is a methyl group, an ethyl group, a propyl group, a butylgroup, a pentyl group, and the like. Moreover, preferred ALA derivativesalso include compounds where the combination of the above R¹ and R² iseach combination of (formyl and methyl), (acetyl and methyl), (propionyland methyl), (butyryl and methyl), (formyl and ethyl), (acetyl andethyl), (propionyl and ethyl), and (butyryl and ethyl).

Among ALAs, a salt of ALA or a derivative thereof can include apharmaceutically acceptable acid addition salt, a metal salt, anammonium salt, an organic amine addition salt, and the like. Acidaddition salts can be exemplified by e.g. each of inorganic acid saltssuch as a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, aphosphate salt, a nitrate salt, and a sulfate salt, and each of organicacid addition salts such as a formate salt, an acetate salt, apropionate salt, a toluenesulfate salt, a succinate salt, an oxalatesalt, a lactate salt, a tartrate salt, a glycolate salt, amethanesulfonate salt, a butyrate salt, a valerate salt, a citrate salt,a fumarate salt, a maleate salt, and a malate salt. Metal salts can beexemplified by each of alkali metal salts such as a lithium salt, asodium salt, and a potassium salt, each of alkaline earth metal saltssuch as a magnesium salt and a calcium salt, and each of metal saltssuch as aluminum and zinc. Ammonium salts can be exemplified by e.g.ammonium salts and alkyl ammonium salts such as a tetramethylammoniumsalt. Organic amine salts can be exemplified by each of salts such as atriethylamine salt, a piperidine salt, a morpholine salt, and atoluidine salt. Note that these salts can also be employed as a solutionat the time of use.

ALAs esters can include, but are not limited to, methyl esters, ethylesters, propyl esters, butyl esters, pentyl esters, and the like.

Among the above ALAs, the most favorable are ALA and various esters suchas an ALA methyl ester, an ALA ethyl ester, an ALA propyl ester, an ALAbutyl ester, and an ALA pentyl ester, as well as hydrochloride salts,phosphate salts, and sulfate salts thereof. In particular, ALAhydrochloride salts and ALA phosphate salts can be exemplified asparticularly favorable.

The above ALAs can be manufactured by e.g. well-known methods such aschemical synthesis, production by microorganisms, and production byenzymes. Moreover, the above ALAs may also forma hydrate or a solvate,and ALAs can be employed alone or in an appropriate combination of twoor more.

When the above ALAs are to be prepared as an aqueous solution, attentionmust be paid so that the aqueous solution will not become alkaline inorder to prevent degradation of ALAs. In the case it becomes alkaline,degradation can be prevented by removing oxygen.

In one embodiment, in the pharmaceutical composition of the presentinvention, one or more types of metal-containing compounds are used incombination. Accordingly, the pharmaceutical composition of the presentinvention can further contain one or more types of metal-containingcompounds. The metal portion of such metal-containing compound caninclude iron, magnesium, zinc, nickel, vanadium, cobalt, copper,chromium, and molybdenum. In a preferred embodiment, the metal portionof the metal-containing compound is preferably iron, magnesium, or zinc,in particular iron.

In the present invention, the iron compound may be an organic salt or aninorganic salt. Inorganic salts can include ferric chloride, ironsesquioxide, iron sulfate, and ferrous pyrophosphate. Organic salts caninclude carboxylic salts such as a hydroxycarboxylic salt, citrate saltssuch as ferrous citrate, iron sodium citrate, sodium ferrous citrate(SFC), and iron ammonium citrate, organic acid salts such as ferricpyrophosphate, heme iron, iron dextran, iron lactate, ferrous gluconate,iron sodium diethylenetriaminepentaacetate, iron ammoniumdiethylenetriaminepentaacetate, iron sodium ethylenediaminetetraacetate,iron ammonium ethylenediaminepentaacetate, iron sodiumdicarboxymethylglutamate, iron ammonium dicarboxymethylglutamate,ferrous fumarate, iron acetate, iron oxalate, ferrous succinate, andiron sodium succinate citrate, as well as triethylenetetramine iron,lactoferrin iron, transferrin iron, sodium iron chlorophyllin, ferritiniron, saccharated iron oxide, and ferrous glycine sulfate.

In the present invention, the magnesium compound can include magnesiumcitrate, magnesium benzoate, magnesium acetate, magnesium oxide,magnesium chloride, magnesium hydroxide, magnesium carbonate, magnesiumsulfate, magnesium silicate, magnesium nitrate, magnesium diammoniumdiethylenetriaminepentaacetate, magnesium disodiumethylenediaminetetraacetate, and magnesium protoporphyrin.

In the present invention, the zinc compound can include zinc chloride,zinc oxide, zinc nitrate, zinc carbonate, zinc sulfate, zinc diammoniumdiethylenetriaminepentaacetate, zinc disodiumethylenediaminetetraacetate, zinc protoporphyrin, and zinc-containingyeast.

The dosage of the metal-containing compound to a subject may be 0-100folds by molar ratio to the dosage of ALA to the subject, desirably0.01-10 folds, and more desirably 0.1-8 folds.

ALAs and the metal-containing compound contained in the pharmaceuticalcomposition of the present invention can be administered as acomposition comprising ALAs and the metal-containing compound or eachcan be administered alone, although it is preferred that even whenadministering each alone, they are administered at the same time. Sametime here means not only administering simultaneously, but also even ifnot simultaneously, administering without substantial interval betweeneach other so that the administration of ALAs and the metal-containingcompound can exert additive effect, preferably synergistic effect.

The administration route of ALAs and the metal-containing compound inthe present invention is not limited, and may be systemic administrationor local administration. Administration routes can include, for example,oral administration including sublingual administration, or parenteraladministration such as inhalation administration, intravenousadministration including infusion, transdermal administration by e.g.patches, suppository, or administration by forced enteral nutritionemploying nasogastric tube, nasointestinal tube, gastrostomy tube, orenterostomy tube. Moreover, as described above, ALAs and themetal-containing compound may be administered from separate routes.

The dosage form of the pharmaceutical composition of the presentinvention may be appropriately determined depending on the saidadministration route, and can include, but is not limited to,injections, infusions, tablets, capsules, fine granules, powders,liquids, solutions dissolved in syrups etc., patches, suppositories, andthe like.

Other optional ingredients such as other medicinal ingredients,nutrients, and carriers can be added as necessary to the pharmaceuticalcomposition according to the present invention. For example, as optionalingredients, various compounding ingredients for preparation of drugssuch as pharmaceutically acceptable ordinary carriers e.g. crystallinecellulose, gelatin, lactose, starch, magnesium stearate, talc, vegetableand animal fat, oil, gum, and polyalkylene glycol, binders, stabilizers,solvents, dispersion mediums, expanders, excipients, diluents, pHbuffers, disintegrants, solubilizers, solubilizing agents, and isotonicagents.

The administration subject of the pharmaceutical composition of thepresent invention is a subject to which an immune checkpoint inhibitoris administered or being administered, typically a subject sufferingfrom cancer. The type of cancer to be the therapeutic subject may changedepending on the immune checkpoint inhibitor used.

The dosage of ALAs to be administered by the pharmaceutical compositionof the present invention, depending on the height, weight, age, and thesymptom of the subject, can be administered in the range of 1 mg-1,000mg, preferably 5 mg-100 mg, more preferably 10 mg-30 mg, furtherpreferably 15 mg-25 mg per kg of subject body weight, when convertedinto ALA (i.e. converted into mass of when R¹ and R² are hydrogen atomsin Formula (I)).

Appropriate number of administrations and the administration frequencyfor the pharmaceutical composition of the present invention can besuitably determined by those skilled in the art in consideration of theadministration conditions of the immune checkpoint inhibitor used incombination (the administration interval, the number of administrations,and the duration of administration). In one embodiment of the presentinvention, the pharmaceutical composition of the present invention isadministered every day from before administration of, at the time ofadministration of, or after administration of the immune checkpointinhibitor.

In the present invention, the effective amount of each of thepharmaceutical composition of the present invention and the immunecheckpoint inhibitor can be administered to a subject at the same timeor at different times, continuously or with intervals. Thepharmaceutical composition of the present invention and the immunecheckpoint inhibitor may be administered to a tumor patient in the sameadministration cycle, or each may be administered in differentadministration cycles. In one embodiment of the present invention, eachof the pharmaceutical composition of the present invention and theimmune checkpoint inhibitor are administered in different administrationcycles.

In one embodiment of the present invention, the administration of thepharmaceutical composition of the present invention to a subject isinitiated before the administration of the immune checkpoint inhibitoris initiated. For example, the pharmaceutical composition of the presentinvention can be administered every day from one week before theadministration of the immune checkpoint inhibitor is initiated.

In another embodiment of the present invention, the administration ofthe pharmaceutical composition of the present invention to a subject isinitiated on the same day as the administration of the immune checkpointinhibitor. For example, the pharmaceutical composition of the presentinvention can be administered every day from the day the administrationof the immune checkpoint inhibitor is initiated. When the pharmaceuticalcomposition of the present invention and the immune checkpoint inhibitorare simultaneously administered, they may be prepared and administeredas a single formulation, or may be simultaneously administered inseparate administration routes.

In further another embodiment of the present invention, theadministration of the pharmaceutical composition of the presentinvention to a subject is initiated after the administration of theimmune checkpoint inhibitor. For example, the pharmaceutical compositionof the present invention can be administered every day after theadministration of the immune checkpoint inhibitor. As long as theantitumor effect by the immune checkpoint inhibitor can be enhanced, thetime point of initiating the pharmaceutical composition of the presentinvention is not particularly limited, and it is preferred that anextended period of time, for example one month or longer, preferablythree weeks or longer, more preferably two weeks of longer, and morepreferably ten days or longer has not elapsed since the time point ofinitiating the immune checkpoint inhibitor.

The terms used herein, except for those that are particularly defined,are employed for describing particular embodiments, and do not intend tolimit the invention.

Moreover, the term “comprising” as used herein, unless the contentclearly indicates to be understood otherwise, intends the presence ofthe described items (such as components, steps, elements, and numbers),and does not exclude the presence of other items (such as components,steps, elements, and numbers).

Unless otherwise defined, all terms used herein (including technical andscientific terms) have the same meanings as those broadly recognized bythose skilled in the art of the technology to which the presentinvention belongs. The terms used herein, unless explicitly definedotherwise, are to be construed as having meanings consistent with themeanings herein and in related technical fields, and shall not beconstrued as having idealized or excessively formal meanings.

The present invention will now be described in further detail withreference to Examples. However, the present invention can be embodied byvarious aspects, and shall not be construed as being limited to theExamples described herein.

EXAMPLES <Example 1> Enhancement of Antitumor Effect by Combination Useof ALA and PD-L1 Antibody Experimental Method

To C57BL/6 mice, 3×10⁵ of mouse melanoma cell strain B16F10 wereinoculated in the flank, and the tumor was allowed to grow for 28 days.On Day 10 and Day 15 from inoculation, 200 μg/head of anti-mouse PD-L1antibody (Clone: MIH5) was intraperitoneally administered for the PD-L1antibody only administration group, the anti-PD-L1 antibody+ALAadministration group, and the anti-PD-L1 antibody+ALA+SFC administrationgroup (FIG. 1). For the anti-PD-L1 antibody+ALA+SFC administrationgroup, 100 mg/kg of ALA hydrochloride salt (from neo ALA CO., LTD.) and157 mg/kg of sodium ferrous citrate (SFC; from KOMATSUYA CORPORATION)were orally administered once a day from Day 10 to Day 16 frominoculation. Moreover, for the anti-PD-L1 antibody+ALA administrationgroup, 100 mg/kg of ALA hydrochloride salt (from neo ALA CO., LTD.) wasorally administered once a day from day 10 to day 16 from inoculation.The group with no treatment after inoculation was placed as the controlgroup.

The tumor diameter was measured every two days, and v wherein v=(minoraxis, mm)²×(major axis, mm)/2 was recorded as the tumor volume (mm³).The values of N were 13 mice for the control group (8 mice were alive onDay 16), 10 mice for the anti-PD-L1 antibody only group (9 mice werealive on Day 16), 4 mice for the anti-PD-L1 antibody+ALA administrationgroup (all mice were alive on Day 16), and 9 mice for the anti-PD-L1antibody+ALA+SFC administration group (all mice were alive on Day 16).

Result

On Day 16 from inoculation, the anti-PD-L1 antibody+ALA administrationgroup had clearly smaller tumor volumes compared to the anti-PD-L1antibody only group (FIG. 2). Moreover, the effect by the anti-PD-L1antibody was more significant when ALA was added and SFC was furtheradministrated (the anti-PD-L1 antibody+ALA+SFC administration group).This result indicates that the antitumor effect of the anti-PD-L1antibody is enhanced by the combination use of ALA+SFC, and it isexpected to contribute to further extension of mouse survival rates.

<Example 2> Enhancement of Antitumor Effect by Combination Use of ALAand PD-1 Antibody Experimental Method

To C57BL/6 mice, 3×10⁵ of mouse melanoma cell strain B16F10 wereinoculated in the flank, and the tumor was allowed to grow for 16 days.On Day 8 and Day 12 from inoculation, 200 μg/mouse of anti-mouse PD-1antibody (Clone: RMP1-14) was intraperitoneally administered for thePD-1 antibody only administration group, the anti-PD-1 antibody+ALAadministration group, and the anti-PD-1 antibody+ALA+SFC administrationgroup (FIG. 3). For the anti-PD-1 antibody+ALA+SFC administration group,100 mg/kg of ALA hydrochloride salt (from neo ALA CO., LTD.) and 157mg/kg of sodium ferrous citrate (SFC; from KOMATSUYA CORPORATION) wereorally administered once a day from Day 8 to Day 16 from inoculation.The group with no treatment after inoculation was placed as the controlgroup.

The tumor diameter was measured every two days, and v wherein v=(minoraxis, mm)²×(major axis, mm)/2 was recorded as the tumor volume (mm³).The values of N were 18 mice for the control group (10 mice were aliveon Day 16), 10 mice for the anti-PD-1 antibody only group (all mice werealive on Day 16), and 10 mice for the anti-PD-1 antibody+ALA+SFCadministration group (9 mice were alive on Day 16).

Result

On Day 16 from inoculation, the anti-PD-1 antibody+ALA+SFCadministration group had clearly smaller tumor volumes compared to theanti-PD-1 antibody only group (FIG. 4). This result indicates that theantitumor effect of the anti-PD-1 antibody is enhanced by thecombination use of ALA+SFC, and it is expected to contribute to furtherextension of mouse survival rates.

INDUSTRIAL APPLICABILITY

According to the present invention, a pharmaceutical composition forenhancing the antitumor effect by an immune checkpoint inhibitor isprovided. Accordingly, by using an immune checkpoint inhibitor and thepharmaceutical composition according to the present invention incombination, it is expected to lead to extension of life and increase ofremission rate for cancer patients.

Moreover, since ALAs which are the active ingredient of thepharmaceutical composition according to the present invention is a typeof natural amino acids contained in vivo that exists widely in animals,plants, and fungi etc., it has the advantage of being able to be usedsafely in vivo.

1. A pharmaceutical composition for enhancing the antitumor effect of animmune checkpoint inhibitor, comprising a compound of Formula (I):[Chemical Formula I]R¹—NHCH₂COCH₂CH₂COOR²  (I), wherein R¹ is a hydrogen atom or an acylgroup, and R² is a hydrogen atom, a linear or branched alkyl group, acycloalkyl group, an aryl group, or an aralkyl group, or a salt or anester thereof, wherein the immune cheekpoint inhibitor is an anti-PD-L1antibody or an anti-PD-1 antibody. 2-4. (canceled)
 5. The pharmaceuticalcomposition according to claim 1, wherein the composition isadministered at the same time or at different times as the immunecheckpoint inhibitor.
 6. The pharmaceutical composition according toclaim 1, wherein: R¹ is selected from the group consisting of a hydrogenatom, an alkanoyl group having 1-8 carbons, and an aroyl group having7-14 carbons, and R² is selected from the group consisting of a hydrogenatom, a linear or branched alkyl group having 1-8 carbons, a cycloalkylgroup having 3-8 carbons, an aryl group having 6-14 carbons, and anaralkyl group having 7-15 carbons.
 7. The pharmaceutical compositionaccording to claim 1, wherein R¹ and R² are hydrogen atoms.
 8. Thepharmaceutical composition according to claim 1, further comprising one,or two or more types of metal-containing compounds.
 9. Thepharmaceutical composition according to claim 8, wherein themetal-containing compound is a compound containing iron, magnesium,zinc, nickel, vanadium, copper, chromium, molybdenum, or cobalt.
 10. Thepharmaceutical composition according to claim 9, wherein themetal-containing compound is a compound containing iron, magnesium, orzinc.
 11. The pharmaceutical composition according to claim 10, whereinthe metal-containing compound is a compound containing iron.
 12. Thepharmaceutical composition according to claim 11, wherein said compoundcontaining iron is sodium ferrous citrate.
 13. A method for enhancingantitumor effect of an immune checkpoint inhibitor, the methodcomprising administering a compound of Formula (I):[Chemical Formula I]R¹—NHCH₂COCH₂CH₂COOR²  (I), wherein R¹ is a hydrogen atom or an acylgroup, and R² is a hydrogen atom, a linear or branched alkyl group, acycloalkyl group, an aryl group, or an aralkyl group or a salt or anester thereof, wherein the immune checkpoint inhibitor is an anti-PD-L1antibody or an anti-PD-1 antibody.
 14. (canceled)